The goal of this supplemental grant application is to establish the zebrafish as a genetic model to elucidate the molecular basis for cell invasion by bacterial toxins. Cholera toxin (CT), the causative agent of Asiatic cholera, will be used to establish the model and to apply a forward genetic screen for toxin-resistant fish. CT typifies the structure and function of the AB5-subunit toxins that enter host cells by traveling backwards on glycolipid receptors associated with lipid rafts from the plasma membrane (PM) to the endoplasmic reticulum (ER). In the ER, a portion of CT unfolds and retro-translocates to the cytosol to cause disease. The molecular mechanisms that underlie retrograde trafficking of membrane glycolipids and retro-translocation of proteins from the ER to cytosol are not fully understood or even completely identified. Our current cell culture systems are not suitable for forward genetics, and the standard genetic systems are not relevant for studies on toxin-induced disease: yeast do not make gangliosides (ganglioside GM1 is the glycolipid receptor for CT), and C. elegans do not make sialic acid (a component of GM1). In mice, the molecular studies that can be applied are inherently biased towards disruption of specific genes and gene products. Thus, there are no established genetic models to study these problems using an unbiased approach. This limits the rate of discovery. Here, we propose that the zebrafish can be used as a genetic model to study all aspects of CT entry into mammalian cells. Such a genetic approach (Aims 1 & 2) is completely novel to the field of toxin biology and microbial pathogenesis, and it may be applied to other equally relevant toxins that enter host cells by other mechanisms (Aim 3). CT and almost all of the toxins to be studied are directly relevant to the NIH program on biodefense (Category A and B). These proposed studies are part of a larger ongoing project in my research group that has been continuously funded by the NIH for the last 15 years. The supplemental funds would provide partial but necessary resources to expand the research program at a critical juncture in the science. Such studies to develop the zebrafish model and to apply a genetic approach to these problems are not funded or even proposed in my current NIH grant. The technology is novel to my laboratory and to the field of bacterial pathogenesis. It will complement the strong biochemical, cellular, and molecular tools already established in my group and successfully applied to the project. The project has direct relevance to the fields of cell biology, microbial pathogenesis, and biodefense, and it has clinical application. [unreadable] [unreadable]